Tracheal antimicrobial peptides, dna sequences and methods for the production and use thereof

ABSTRACT

The present invention provides a new class of polypeptides with antimicrobial activity, termed &#34;tracheal antimicrobial peptides,&#34; cDNA sequences encoding for the peptides and methods for the production and use thereof.

FIELD OF THE INVENTION

The present invention relates generally to antimicrobial peptidesreferred to herein as tracheal antimicrobial peptides. Moreparticularly, the present invention is related to a new class ofpolypeptides with antimicrobial activity, cDNA sequences encoding forthe peptides, methods for the production and use thereof.

BACKGROUND OF THE INVENTION

The respiratory epithelium of mammals is a complex tissue responsiblefor numerous physiological functions, one of which is forming a keybarrier to potentially harmful environmental threats. Multiple defensemechanisms have been identified which protect the respiratory tract frominhaled agents that are known to be responsible for airway disease, suchas infectious agents, gases, and particulates. Newhouse, M.T. and J.Bienenstock, "Respiratory Tract Defense Mechanisms," Textbook ofPulmonary Disease, Little, Brown and Comp (1989). These multipledefenses are the result of a combination of anatomical design of theairway, together with the physiological role of local and circulatingcells.

Recent isolation and characterization of antimicrobial peptides in avariety of species and tissues has unveiled a new component of animalhost defense. These various peptides, which can be classified intofamilies based on common sequences, secondary structure and/or sites ofactivity, are believed to participate in defense against potentialmicrobiological pathogens. Cecropins were the first well characterizedfamily of structurally related antimicrobial peptides and are found in awide distribution of insects. Boman, H.G. and D. Hultmark, Ann. Rev.Microbiol., 41:103-126 (1987). They are coordinately expressed in thefat body of insect larvae following infection or injury. In vertebrates,the magainin family of antimicrobial peptides have been isolated fromglands of the skin and gastrointestinal tract of Xenopus Iaevis, and arethought to form the basis for a defense system of the amphibian mucosalsurfaces against infection. Soravia, E., G. Martini et al.,"Antimicrobial properties of peptides from Xenopus granular glandsecretions," FEBS Lett., 228:337-40 (1988); Zasloff, M.A., "Magainins, aclass of antimicrobial peptides from Xenopus skin: Isolation,characterization of two active froma, and partial cDNA sequence of aprecursor," Proc Natl Acad Sci USA, 84:5449-53 (1987). Defensins arepeptides found in phagocytic cells isolated from several mammalianspecies including man, and may be characterized by 8 invariant residueswithin the sequence. Gabay, J.E., "Microbicidal mechanisms ofphagocytes," Curr Opin Immunol, 1(1):36-40 (1988); Gabay, J.E., R.W.Scott et al., "Antibiotic proteins of human polymorphonuclearleukocytes," Proc Natl Acad Sci USA, 86(14):5610-4 (1989); Ganz, T.,"Extracellular release of antimicrobial defensins by humanpolymorphonuclear leukocytes," Infect Immun, 55(3):568-71 (1987): Ganz,T., J.A. Metcalf et al., "Microbicidal/cytotoxic proteins of neutrophilsare deficient in two disorders: Chediak-Higashi syndrome and `specific`granule deficiency," J CIin Invest, 82(2):552-6 (1988); Ganz, T., J.R.Rayner et al., "The structure of the rabbit macrophage defensin genesand their organ-specific expression," J Immunol, 143(4):1358-65 (1989);Ganz, T., M.E. Selsted et al., "Antimicrobial activity of phagocytegranule proteins," Semin Respir Infect, 1(2):107-17 (1986); Ganz, T.,M.E. Selsted et al., "Defensins," Eur J Hematol, 44(1):1-8 (1990a);Ganz, T., M.E. Selsted et al., "Defensins," Eur J Haematol, 44(1):1-8(1990b); Ganz, T., M.E. Selsted et al., "Defensins. Natural peptideantibodies of human neutrophils," J Clin Invest, 76(4):1427-35 (1985).They possess antimicrobial activity in vitro against bacteria, fungi,and viruses, and may contribute to the "oxygen-independent" defensepathways of these cells. Lehrer, R.I., T. Ganz et al.,"Oxygen-independent bactericidal systems. Mechanisms and disorders,"Hematol Oncol Clin North Am, 2(1):159-69 (1988). Expression of defensinin a non-myeloid tissue source, the mouse small intestinal crypt cells,has also been reported. Ouellette, A.J., R.M. Greco et al.,"Developmental regulation of cryptdin, a corticostatin/defensinprecursor mRNA in mouse small intestinal crypt epithelium," J Cell Biol,108(5):1687-95 (1989).

Cecropins, magainins, and defensins all share the properties of beingcationic and membrane active, and evidence suggests that theirantimicrobial activity is secondary to their ability to selectivelydisrupt membranes, possibly by channel formation. Bevins, C.L. and M.A.Zasloff, "Peptides from frog skin," Ann. Rev. Biochem., 59:395-414(1990); Kagan, B.L., M.E. Selsted et al., "Antimicrobial defensinpeptides form voltage-dependent ion-permeable channels in planar lipidbilayer membranes," Proc Natl Acad Sci USA, 87(1):210-4 (1990); Lehrer,R.I., A. Barton et al., "Interaction of human defensins with Escherichiacoli. Mechanism of bactericidial activity," J Clin Invest, 84(2):553-61(1989); Zasloff, M.A., "Magainins, a class of antimicrobial peptidesfrom Xenopus skin: Isolation, characterization of two active froma, andpartial cDNA sequence of a precursor," Proc Natl. Acad Sci USA,84:5449-53 (1987).

These newly emerging family of basic, cysteine-rich peptides withantimicrobial activity found throughout the animal kingdom include thedefensins (Ganz, T., M.E. Selsted et al , "Defensins," Eur J Haematol,44(1): 1-8 (1990b)), insect defensins (Lambert, J., E. Keppi et al.,"Insect immunity:isolation from immune blood of the dipteran Phormiaterranovae of two insect antibacterial peptides with sequence homologyto rabbit lung macrophage bactericidal peptides" [published erratumappears in Proc Natl Acad Sci USA May;86(9):3321(1989)]. Proc Natl AcadSci USA 86(1): 262-6 (1989)), bactenecins (Romeo D., B. Skerlavaj etal., "Structure and bactericidal activity of an antibiotic dodecapeptidepurified from bovine neutrophils," J Biol Chem, 263:9573-75 (1988)),sapecins (Matsuyama, K. and Natori, S. "Purification of threeantibacterial proteins from the culture medium of NIH-Sape-4, anembryonic cell line of Saroophaga peregrina,"J biol Chem, 263:17112-16(1988)) and royalisin (Fujiwara, S., J. Imai et al., " A potentantimicrobial protein in royal jelly," J Biol Chem, 265: 11333-37(1990)). Defensins are basic peptides of 30 to 34 amino acids with 3disulfide bonds. The known characterized defensins from both myeloid andnon-myeloid tissues all have highly conserved amino acid residues withinthe family, including 6 invariant cysteines. Aside from a pair ofcysteine residues near the carboxy-terminus of the trachealantimicrobial peptide of this invention, no consensus or other residuesare shared between these peptides. Furthermore, the 5' region of allknown defensin cDNAs are strikingly conserved even across species, andno similarity with this consensus region is found in the trachealantibiotic peptide's cDNA. Comparison with the other cysteine containingantimicrobial peptides shows no similarity.

Formal searches of the NRBF protein data base using a modification (IBI)of fastP, Lipmann, D.J. and W.R. Pearson, "Rapid and sensitive proteinsimilarity searches,⃡ Science, 227:1435-41 (1985) and the IntelligeneticsSearch found no protein sequences that disclose the trachealantimicrobial peptides of the instant invention. A nucleotide-basedsearch of the GenBank data base using the University of WisconsinGenetics analysis software, Devereux, J., P. Haeberli et al., "Acomprehensive set of sequence analysis programs for the VAX," Nucl.Acids Res., 12:387-95 (1984), was similarly unrevealing.

SUMMARY OF THE INVENTION

There is provided by this invention a novel tracheal antimicrobialpeptide (TAP) and a novel precursor of TAP.

There is provided by this invention a novel cDNA coding for a TAP and anovel cDNA coding for a TAP precursor.

There is provided by this invention a novel protein produced from thecDNA coding for a TAP and a novel protein produced from the cDNA codingfor a TAP precursor.

There is provided by this invention a novel substantially purified,isolated mammalian TAP precursor comprising a protein having at least inpart or in whole substantially the same amino acid sequence as theprotein defined in SEQ ID NO:3.

There is provided by the invention a substantially purified, isolatedmammalian TAP comprising a protein having at least in part or in wholesubstantially the same amino acid sequence and at least the sameantimicrobial activity as the protein defined in SEQ ID NO:1.

There are provided novel methods of use and diagnosis for TAP and cDNAcoding for TAP.

There are provided novel recombinant host cells transformed with DNAcoding for TAP or a portion thereof sufficient for the expression of TAPby said host cells.

There is provided a novel method of producing TAP which comprisesculturing recombinant host cells wherein a recombinant DNA transformedin said host cell has a DNA sequence encoding TAP, operably linked toappropriate regulatory control sequences which are capable of effectingthe expression of said coding sequences in said transformed cells.

There is provided a novel recombinant vector capable of expression in asuitable expression system which comprises a DNA sequence encoding TAPoperably linked to control sequences compatible with said expressionsystem.

Applicants have found that the ciliated respiratory mucosa of mammalscontains peptide-based antimicrobial activity, to complement otherdefense systems of the airway. An abundant novel peptide found inextracts of the mammalian tracheal mucosa, is isolated herein on thebasis of potent antimicrobial activity. This molecule is hereinafterreferred to as tracheal antimicrobial peptide (TAP).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Purification of the antimicrobial peptide from Bovine trachea:

A. P-30 gel filtration chromatogram; fractions containing antimicrobialactivity are marked.

B. Antimicrobial plate assay of P-30 fractions. 2μl of each fraction wasspotted on a lawn of E. coli strain D31 and incubated overnight at 37°C.

C. Ion exchange HPLC chromatogram of P-30 15 antimicrobial fractions.Antimicrobial fraction eluting at 26 minutes is marked with an arrow.

D. Reverse phase HPLC chromatogram of the antimicrobial fraction fromion exchange HPLC.

FIG. 2. Amino acid sequence of TAP and related nucleotide sequences:

A. Amino acid sequence (SEQ ID NO:1) of a TAP based on a combination ofpeptide amino acid sequence analysis, mass spectral analysis, and cDNAsequence analysis. The arrows indicate the results from Edmandegradation analysis: sequence 1 is from direct amino-terminal analysis,sequence 2 is following cyanogen bromide cleavage and HPLC purification.Cysteine residue were determined following reduction and treatment with4-vinylpyridine.

B Nucleotide sequences (SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:12) ofdegenerate oligonucleotides used for PCR amplification. A 1:1 mixture ofoligonucleotides was used as an upstream primer. Abbreviations used:Y=C,T; R=A,G; D=G,A,T; B=C,G,T. Lower case letters denote 5' flankingsequences included in the oligonucleotide to aid subcloning.

C. Nucleotide sequence of oligonucleotides (SEQ ID NO:8 and SEQ ID NO:9)used in cDNA screening. Selection of sequence was based on partialpeptide sequence and codon usage tables.

D. Partial nucleotide sequence (SEQ ID NO:2) of bovine TAP cDNA obtainedfrom dideoxy-sequence analysis.

FIG. 3. Nucleotide sequence (SEQ ID NO:4) and predicted amino acidsequence (SEQ ID NO:3) of a TAP precursor. Amino acid numbering startswith the initiation codon. The mature peptide (SEQ ID NO:1) beginning atresidue 27 is underlined. The polyadenylation signal is boxed.

FIG. 4. Northern blot analysis of TAP (SEQ ID NO:1) message. Lane 1,10μg total bovine trachea RNA; lane 2, 10μg total bovine lung RNA.

DETAILED DESCRIPTION OF THE INVENTION

The term "antimicrobial" as used herein refers to killing microorganismsor suppressing their multiplication and growth.

As used herein, "TAP" refers to a protein having at least in part or inwhole substantially the same amino acid sequence and at least the sameantimicrobial activity as the protein defined in SEQ ID NO:1. Saidantimicrobial activity being defined as substantially preventing thegrowth of E. coli K12 strain D31 under the conditions described below(TableII) at a concentration of at least 100 μg/ml TAP. For convenience,a protein having at least in part or in whole substantially the sameamino acid sequence as the protein defined in SEQUENCE ID NO:3 ishereinafter referred to as a TAP precursor.

The TAP protein of the invention, depending on the pH of itsenvironment, if suspended or in solution, or of its environment whencrystallized or precipitated, if in solid form, may be in the form ofpharmaceutically acceptable salts or may be in neutral form. The freeamino groups of the protein are, of course, capable of forming acidaddition salts with, for example, organic acids such as hydrochloric,phosphoric, or sulfuric acid; or with organic acids such as, forexample, acetic, glycolic, succinic, or mandelic acid. The free carboxylgroups are capable of forming salts with bases, including inorganicbases such as sodium, potassium, or calcium hydroxides, and such organicbases as piperidine, glucosamine, trimethylamine, choline, and caffeine.In addition, the protein may be modified by combination with otherbiological materials such as lipids and saccharides, or by side chainmodification such as acetylation of amino groups, phosphorylation ofhydroxyl side chains, or oxidation of sulfhydryl groups.

Modifications of TAP are included within the scope of the definition, solong as the antimicrobial activity as described herein is retained.Finally, it is understood that minor modifications of TAP may result inproteins which have substantially equivalent or enhanced antimicrobialactivity as compared to the sequence set forth in SEQ ID NO:I. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental such a through mutation in hosts which are TAPproducers. All of these modifications are included as long as theantimicrobial activity, is retained.

Acid extracts of mammalian tracheal mucosa, e.g. bovine, have been foundto have the peptide TAP in abundant quantities, a peptide with potentantimicrobial activity (FIG. 1). A mammalian TAP (SEQ ID NO:1) wasisolated by a combination of size-exclusion (FIG. 1A), ion-exchange(FIG. 1C), and reverse-phase (FIG. 1D) chromatographic fractionationsusing antimicrobial activity against a strain of E. coli as a functionalassay. Purity of the isolated peptide (SEQ ID NO:1) was >95% asdetermined by a combination of analytical reverse-phase andcapillary-gel electrophoresis (data not shown). The yield of theisolated material was 2 μg/g of wet tracheal mucosa.

The mammalian TAP, SEQ ID NO:1, was characterized by amino acid sequence(data not shown), and compositional analysis (Table I) Mass spectralanalysis determined the molecular weight of the peptide (SEQ ID NO:1) tobe 4085 Da.

A cDNA (SEQ ID NO:4) corresponding to the precursor peptide, was cloned(FIG. 3), and contains an open reading frame of 64 amino acids (SEQ IDNO:3). It is believed that the thirty-eight carboxy-terminal residues ofthis open reading frame correspond to the isolated peptide (SEQ ID NO:1)based on several observations. First, the 33 amino acids determined fromthe amino acid sequence data align perfectly with residues 26-59 of thededuced sequence (FIG. 2A (SEQ ID NO:1) v. 2D (SEQ ID NO:2)). Second,the amino acid composition agrees favorably with the deduced amino acidsequence (Table I). Finally, the observed molecular ion of the isolatedprotein (SEQ ID NO:1), 4085 Da is in complete agreement with the deducedsequence, assuming the six cysteine residues all participate inintramolecular disulfide bonds. The predicted pI is 13.0, and there areno aromatic residues, both consistent with the observed protein data.

When assayed in vitro against several different strains of microbes,including some which are respiratory pathogens, a mammalian TAP (SEQ IDNO:1) of bovine origin showed similar inhibitory activity to that ofsynthetic magainin 2-NH₂ (SEQ ID NO:5) , a naturally occurringantibiotic peptide from frog skin. See TableII. While TAP (SEQ ID NO:1)was most active against E. coli and K. pneumonia, significantantimicrobial activity was also seen when applied to C. albicans, thusTAP's spectrum of activity is believed to span at least to both bacteriaand fungi.

Having described the amino acid sequence of a TAP (SEQ ID NO:1),including the precursor peptide (SEQ ID NO:3), it is believed thesepolypeptides can be routinely synthesized in substantially pure form bystandard techniques well known in the art, such as commerciallyavailable peptide synthesizers and the like.

Additionally, it is believed TAP can be efficiently prepared using anyof numerous well known recombinant techniques such as those described inU.S. Pat. No. 4,677,063 which patent is incorporated by reference as iffully set forth herein. Briefly, most of the techniques which are usedto transform cells, construct vectors, extract messenger RNA, preparecDNA libraries, and the like are widely practiced in the art, and mostpractitioners are familiar with the standard resource materials whichdescribe specific conditions and procedures. However, for convenience,the following paragraphs may serve as a guideline.

Procaryotes most frequently are represented by various strains of E.coli. However, other microbial strains may also be used, such asbacilli, for example Bacillus subtilis, various species of Pseudomonas,or other bacterial strains. In such procaryotic systems, plasmid vectorswhich contain replication sites and control sequences derived from aspecies compatible with the host are used. For example, E. coli istypically transformed using derivatives of pBR322, a plasmid derivedfrom an E. coli species by Bolivar, et al, Gene (1977) 2:95. pBR322contains genes for ampicillin and tetracycline resistance, and thusprovides additional markers which can be either retained or destroyed inconstructing the desired vector. Commonly used procaryotic controlsequences include promoters for transcription initiation, optionallywith an operator, along with ribosome binding site sequences, includesuch commonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems (Chang, et al., Nature (1977) 198:1056)and the tryptophan (trp) promoter system (Goeddel, et al. Nucleic AcidsRes (1980) 8:4057) and the lambda derived P_(L) promoter and N-generibosome binding site (Shimatake, et al., Nature (1981) 292:128).

In addition to bacteria, eucaryotic microbes, such as yeast, may also beused as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker'syeast, are most used although a number of other strains are commonlyavailable. While vectors employing the 2 micron origin of replicationare illustrated, Broach, J.R., Meth Enz (1983) 101:307, other plasmidvectors suitable for yeast expression are known (see, for example,Stinchcomb, et al., Nature (1979) 282:39, Tschempe, et al., Gene(1980)10:157 and Clark, L., et al., nz (1983) 101:300). Controlsequences for yeast vectors include promoters for the synthesis ofglycolytic enzymes (Hess, et al., J Adv Enzyme Req (1968) 7:149;Holland, et al. Biochemistry (1978) 17:4900). Additional promoters knownin the art include the promoter for 3-phosphoglycerate kinase (Hitzeman,et al., J Biol Chem (1980) 255:2073), and those for other glycolyticenzymes such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Other promoters,which have the additional advantage of transcription controlled bygrowth conditions are the promoter regions for alcohol dehydrogenase 2,isocytochrome C, acidphosphatase, degradative enzymes associated withnitrogen metabolism, and enzymes responsible for maltose and galactoseutilization (Holland, ibid). It is also believed terminator sequencesare desirable at the 3' end of the coding sequences. Such terminatorsare found in the 3' untranslated region following the coding sequencesin yeast-derived genes. Many of the vectors illustrated contain controlsequences derived from the enolase gene containing plasmid peno46(Holland, M.J., et al., J Biol Chem (1981) 256:1385) or the LEU2 geneobtained from YEp13 (Broach, J., et al., Gene (1978) 8:121), however anyvector containing a yeast compatible promoter, origin of replication andother control sequences is suitable.

It is also, of course, possible to express genes encoding polypeptidesin eucaryotic host cell cultures derived from multicellular organisms.See, for example, Tissue Cultures, Academic Press, Cruz and Patterson,editors (1973). Useful host cell lines include VERO, HeLa cells, andChinese hamster ovary (CHO) cells. Expression vectors for such cellsordinarily include promoters and control sequences compatible withmammalian cells such as, for example, the commonly used early and latepromoters from Simian Virus 40 (SV 40) Fiers, et al., Nature (1978)273:113), or other viral promoters such as those derived from polyoma,Adenovirus 2, bovine papilloma virus, or avian sarcoma viruses. Generalaspects of mammalian cell host system transformations have beendescribed e.g. by Axel; U.S. Pat. No. 4,399,216. It now appears, alsothat "enhancer" regions are important in optimizing expression; theseare, generally, sequences found upstream or downstream of the promoterregion in non-coding DNA regions. Origins of replication may beobtained, if needed, from viral sources. However, integration into thechromosome is a common mechanism for DNA replication in eucaryotes.Plant cells are also now available as hosts, and control sequencescompatible with plant cells such as the nopaline synthase promoter andpolyadenylation signal sequences (Depicker, A., et al., J Mol Appl Gen(1982) 1:561) are available.

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described by Cohen, S.N., Proc Natl Acad Sci (USA)(1972) 69:2110, or methods described in Molecular Cloning: A LaboratoryManual (1988) Cold Spring Harbor Press, could be used for procaryotes orother cells which contain substantial cell wall barriers. Infection withAgrobacterium tumefaciens (Shaw, C. H., et al., Gene (1983) 23:315) isbelieved useful for certain plant cells. For mammalian cells withoutsuch cell walls, the calcium phosphate precipitation method of Grahamand van der Eb, Virology (1978) 52:546 can be used. Transformations intoyeast can be carried out according to the method of Van Solingen, P., etal., J Bact (1977) 130:946 and Hsiao, C.L., et al., Broc Natl Acad Sci(USA) (1979) 76:3829.

cDNA or genomic libraries can be screened using the colony hybridizationprocedure. Generally, each microtiter plate is replicated onto duplicatenitrocellulose filter papers (S&S type BA-85) and colonies are allowedto grow at 37° C. for 14-16 hr on L agar containing 50 μg/ml Amp. Thecolonies are lysed and DNA fixed to the filter by sequential treatmentfor 5 min with 500 mM NaOH, 1.5M NaCl, and are washed twice for 5 mineach time with 5xstandard saline citrate (SSC). Filters are air driedand baked at 80° C. for 2 hr. The duplicate filters are prehybridized at42° C. for 6-8 hr with 10 ml per filter of DNA hybridization buffer(5xSSC, pH 7.0 5×Denhardt's solution (polyvinylpyrrolidine, plus Ficolland bovine serum albumin; 1×=0.02% of each), 50 mM sodium phosphatebuffer at pH 7.0, 0.2% SDS, 20 μg/ml Poly U, and 50 μ g/ml denaturedsalmon sperm DNA).

The samples can be hybridized with kinased probe under conditions whichdepend on the stringency desired. Typical moderately stringentconditions employ a temperature of 42° C. for 24-36 hr with 1-5ml/filter of DNA hybridization buffer containing probe. For higherstringencies high temperatures and shorter times are employed.Generally, the filters are washed four times for 30 min each time at 37°C. with 2×SSC, 0.2% SDS and 50 mM sodium phosphate buffer at pH 7, thenare washed twice with 2×SSC and 0.2% SDS, air dried, and areautoradiographed at -70° C. for 2 to 3 days.

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniqueswhich are well understood in the art. Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and religated inthe form desired.

Site specific DNA cleavage can be performed by treating the DNA with asuitable restriction enzyme (or enzymes) under conditions which aregenerally understood in the art, and the particulars of which arespecified by the manufacturer of these commercially availablerestriction enzymes. See, e.g., New England Biolabs, Product Catalog. Ingeneral, about 1 μg of plasmid or DNA sequence is cleaved by one unit ofenzyme in about 20 μl of buffer solution. Incubation times of about onehour to two hours at about 37° C. are workable, although variations canbe tolerated. After each incubation, protein can be removed byextraction with phenol/chloroform, and may be followed by etherextraction, and the nucleic acid recovered from aqueous fractions byprecipitation with ethanol followed by running over a Sephadex G-5 spincolumn. If desired, size separation of the cleaved fragments may beperformed by polyacrylamide gel or agarose gel electrophoresis usingstandard techniques. A general description of size separations can befound in Methods in Enzymology (1980) 65:499-560.

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 min at 20° to 25° C. in 50 mM Tris pH 7.6, 50 mM NaCl, 6mM MgCl,, 6 mM DTT and 5-10 μM dNTPs. The Klenow fragment fills in at 5'sticky ends but chews back protruding 3' single strands, even though thefour dNTPs are present. If desired, selective repair can be performed bysupplying only one of the, or selected, dNTPs within the limitationsdictated by the nature of the sticky ends. After treatment with Klenow,the mixture is extracted with phenol/chloroform and ethanol precipitatedfollowed by running over a Sephadex G-50 spin column. Treatment underappropriate conditions with Sl nuclease results in hydrolysis of anysingle-stranded portion.

Synthetic oligonucleotides can be prepared by the triester method ofMetteucci, et al. (J Am Chem Soc (1981) 103:3185) or using commerciallyavailable automated oligonucleotide synthesizers. Kinasing of singlestrands prior to annealing or for labeling is achieved using an excess,e.g., approximately 10 units of polynucleotide kinase to 0.1 nmolesubstrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl:, 5 mMdithiothreitol, 1-2 Mm ATP, 1.7 pmoles γ³² P-ATP (2.9 mCi/mmole), 0.1 mMspermidine, 0.1 mM EDTA.

Ligations can be performed in 15-30 μl volumes under the followingstandard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl,,10 mM DTT, 33 μg/ml GSA, 10 mM-50 mM NaCl, and either 40 μM ATP,0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for "sticky end"ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C.(for "blunt end" ligation). Intermolecular "sticky end" ligations areusually performed at 33-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). Intermolecular blunt end ligations (usuallyemploying a 10- 30 fold molar excess of linkers) are performed at 1 μMtotal ends concentration.

In vector construction employing "vector fragments", the vector fragmentcan be treated with bacterial alkaline phosphatase (BAP) in order toremove the 5' phosphate and prevent religation of the vector. BAPdigestions can be conducted at pH 8 in approximately 150 mM Tris, in thepresence of Na+ and Mg+² using about 1 unit of BAP per μg of vector at60° C. for about one hour. In order to recover the nucleic acidfragments, the preparation is extracted with phenol/chloroform andethanol precipitated and desalted by application to a Sephadex G-50 spincolumn. Alternatively, religation can be prevented in vectors which havebeen double digested by additional restriction enzyme digestion of theunwanted fragments.

For portions of vectors derived from cDNA or genomic DNA which requiresequence modifications, site specific primer directed mutagenesis can beused. This is conducted using a primer synthetic oligonucleotidecomplementary to a single stranded phage DNA to be mutagenized exceptfor limited mismatching, representing the desired mutation. Briefly, thesynthetic oligonucleotide is used as a primer to direct synthesis of astrand complementary to the phage, and the resulting double-stranded DNAis transformed into a phage-supporting host bacterium. Cultures of thetransformed bacteria are plated in top agar, permitting plaque formationfrom single cells which harbor the phage.

Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form; 50% will have the original sequence.The resulting plaques can be hybridized with kinased synthetic primer ata temperature which permits hybridization of an exact match, but atwhich the mismatches with the original strand are sufficient to preventhybridization. Plaques which hybridize with the probe are then picked,cultured, and the DNA recovered.

Correct ligations for plasmid construction can be confirmed by firsttransforming a suitable host with the ligation mixture. Successfultransformants are selected by ampicillin, tetracycline or otherantibiotic resistance or using other markers depending on the mode ofplasmid construction, as is understood in the art. Plasmids from thetransformants can then be prepared according to the method of Clewell,D.B., et al. Proc Natl Acad Sci (USA) (1969) 62:1159, optionallyfollowing chloramphenicol amplification (Clewell, D.B., J Bacteriol(1972) 110:667). The isolated DNA Is analyzed by restriction and/orsequenced by the dideoxy method of Snager, F., et al. Proc Natl Acad Sci(USA) (1977) 74:5463 as further described by Messing, et al., F. Supp.Nucleic Acids Res (1981) 9.309, or by the method of Maxam, et al.,Methods in Enzymology (1980) 65:499.

In a third alternative, TAP can be prepared directly from a mammaliantrachea as described below.

Also provided by this invention are methods of treating a microbialinfection, such as bacterial or fungal, comprising administering to amammal in need of such treatment an antimicrobially effective amount ofTAP. Such treatment can be systemic or topical for treatment of e.g.acne, burns, eye infections, mouthwash, deodorant or topical fungicide.TAP could also be used as a contact disinfectant.

For use as an antimicrobial agent, TAP can be formulated intopharmacological compositions containing an effective amount of TAP and ausual nontoxic carrier, such carriers being known to those skilled inthe art. The composition can be given via a route of administrationsuited to the form of the composition. Such compositions are, forexample, in the form of usual liquid preparations including solutions,suspensions, emulsions and the like which can be given orally,intravenously, subcutaneously or intramuscularly. The composition can beadministered in an antimicrobially effective amount, generally a dose ofabout 0.1 to about 100 mg/kg/day, calculated as protein is expected tobe useful.

Other utilities of the invention disclosed herein include, use of thecDNA sequence of TAP as a marker for various genetic studies in themammal. This type of marker can be used to diagnose genetic diseaseswhich may be linked to this marker, if not directly due to a defect inthe TAP gene. In addition, genetic markers such as this can be used inRestriction Fragment Length Polymorphism (RFLP) studies for breedingpurposes as are other genetic markers.

Additionally, since TAP is found in the mammalian airway and mayrepresent part of the host defense against infections, it is believedthat overexpression of TAP is induced by the presence of certainmicrobes. Therefore, the presence of higher quantities of TAP mayindicate the presence of specific organisms or the beginning of aninfectious state. Quantitation of TAP protein by immunoassay or othersuch methods well known in the art, or measurement of TAP messenger RNAby well known hybridization techniques may serve as a diagnostic toolfor infections.

MATERIALS AND METHODS General Methodology

All reagents were standard reagent grade from Baker, Philipsburg, NJ orFisher, Pittsburg, PA unless otherwise noted. All bacteriological mediawere from Difco, Detroit, MI. Restriction enzymes were purchased fromBethesda Research Laboratories (Gaithersburg, MD) and were usedaccording to manufacturer's protocol. Oligonucleotide probes were endlabelled to a specific activity of ca. 10⁷ DPM/pmol using gamma-[³²P]dCTP (3000Ci/mmol, DuPont, Wilmington, DE) and T4 polynucleotidekinase (Stratagene, LaJolla, CA). Double-stranded DNA probes werelabelled to a specific activity of ca 10⁹ DPM/μg using alpha-[³² P]dCTP(800Ci/mmol), DuPont, Wilmington, DE) and T7 DNA polymerase with randomoligonucleotide primers (Stratagene). Purified plasmid DNA was sequencedusing the dideoxy-termination method with T7 DNA polymerase (U.S.Biochemicals).

Tissue

A segment of adult bovine trachea (just proximal to the carina andapproximately 40 cm in length) was obtained fresh from a local meatprocessing plant. The tissue was immediately placed on wet ice andprocessed within 2-3 hours. Preliminary experiments indicated thatimmediate processing of tissue gave no significant further improvementin yield. The epithelium and adherent connective tissue, dissected onice from the underlying connective tissue and cartilage, was placedimmediately in liquid nitrogen. The frozen tissue was then stored at-70° C. for periods up to several months before further processing.

Protein Isolation

The frozen tracheal epithelium was pulverized with a mortar and pestleunder liquid nitrogen. The frozen tissue powder was placed in boiling10% (v/v) acetic acid, and boiling was continued for 10 minutes. Thesolution was allowed to cool to room temperature, and centrifuged at23,000×g. for 30 minutes at 10° C. The resulting supernatant was dividedinto 30 ml. aliquots and each aliquot was applied to a C18 Sep-Pakcartridge (Millipore Corp., Bedford, MA). This, and all subsequentprocedures were performed at room temperature. The cartridges werewashed with 0.1% trifluoroacetic acid (TFA) in H₂ O (Buffer C) and theneluted with 4 ml. of acetonitrile/0.1% TFA (60:40, v/v; Buffer D). Thecartridge eluates were dried, resuspended in 1-2 ml 6Mguanidinium-HCl/20mM Tris-HCl, pH 7.4. The solution was applied to aBiogel P-30 column (40 cm.×2.5 cm. diameter, Bio Rad) which had beenpre-equilibrated with 50mM ammonium formate, pH 4.1. The exclusion limitwas 40 kDa. The column was developed with the same ammonium formatebuffer, and each fraction (2 ml.) was lyophilized, resuspended in water(0.1 ml.) and assayed for antimicrobial activity as described. Zasloff,M.A., "Magainins, a class of antimicrobial peptides from Xenopus skin:Isolation, characterization of two active froma, and partial cDNAsequence of a precursor," Proc Natl Acad Sci USA, 84:544953 (1987). Theactive fractions were pooled, and applied to a sulfoethyl ion exchangeHPLC column (Poly LC, Columbia, MD). A 45 minute linear elution-gradientfrom buffer A to buffer B was employed at a flow rate of 1 ml./min.Buffer A contained 25% acetonitrile/5 mM potassium phosphate, pH 5.3,and buffer B was identical to buffer A except that it also contained 1 MNaCl. Preliminary experiments had established that only a fractioneluting at 26 minutes contained significant antimicrobial activity. Insubsequent isolations, this fraction was applied to a reverse-phase HPLCcolumn and fractionated using a linear gradient buffer C to buffer D at1 ml./min. The peak fraction eluting at 28.5 minutes was lyophilized andresuspended in H₂ O at an approximate concentration of 0.5 mg/ml.

Protein Sequence Analysis

The isolated peptide was subjected to amino acid analysis using an aminoacid analyzer with automated hydrolysis (Applied Biosystems model 420,ABS, Foster City, CA). Sequence analysis was determined by the Edmandegradation method on a pulsed liquid phase sequencer (AppliedBiosystems model 477A, ABS). Cysteine residues were identified bysulfhydryl reduction followed by reaction with 4-vinylpyridine, SelstedM.E., S.S. Harwig et al., "Primary structures of three human neutrophildefensins," J Clin Invest, 76(4) 1436-9 (1985), prior to sequenceanalysis. The C-terminal portion of the peptide was isolated by HpLCfollowing cleavage with cyanogen bromide. Matsudaira, P. "LimitedN-Terminal Sequence Analysis," Guide to Protein Purification. Deutschered. Academic Press (1990). Cysteine residues were identified bysulfhydry reduction followed by reaction with 4-vinylpyridine prior tosequence analyses. Wilde, C.G., J.E. Griffith, et al., "Purification andCharacterization of human neutrophil peptide 4, a novel member of thedefensin family," J Biol Chem, 264 19 : 11200-3 1989).

Mass Spectroscopy

Molecular weight of the unreduced peptide was independently determinedby fast-atom bombardment on a JEOL HX110 mass spectrometer at 1000resolution (Structural Biochemistry Center, University of Maryland,Baltimore County) and on a VG analytical ZAB 2-SE high field massspectrometer operating at Vacc=8kv (MScan, Inc., West Chester, PA).

PCR Amplification

The pCR product was obtained using the degenerate oligonucleotide5'-GAGCTCDGTICCDATYTGYTTCAT, SEQ ID NO:7, as an antisense primer, a 1:1mixture of 5'-GAATTCAAYCCHGTBAGiTGYGTT, SEQ ID NO:12, and 5'-GAATTCAAYCCHGTBTCYTGYGTT, SEQ ID NO:6, as sense primers and a pool ofbovine tracheal cDNA (prior to size fractionation) as a template. Thegeneral protocol for polymerase chain reaction (PCR) amplificationSaiki, R.K., D.H. Gelfand et al., "Primer-directed enzymaticamplification of DNA with a Thermostable DNA polymerase," Science,239:487-91 (1988) using reagents from a Gene Amp kit (Cetus) wasmodified for using the degenerate primers: final concentration templatecDNA was 0.2ng/ml and of primers was 1μM. After initial denaturation at94° C., reactions were incubated for 30 cycles of one minute at 94° C.,one minute at 55° C. and three minutes at 72° C. Bands were purified byelectroelution after electrophoresis in polyacrylamide gels.

cDNA cloning

The techniques used for cDNA library construction have been describedand reagents were from Invitrogen, San Diego, CA unless noted otherwise.Total mRNA was isolated from bovine tracheal epithelium tissue usingaccording to the protocol of Chirgwin et al., "Isolation of biologicallyactive ribonucleic acid from sources enriched in ribonuclease,"Biochem., 18:5294-99 (1979). Poly (A+) mRNA was selected using oligo dTcellulose columns (3'→5',Inc.). Poly A enriched RNA from bovine trachealepithelium was reversed transcribed using a commercially available(Invitrogen) oligo dT primer. Second-strand cDNA was synthesized usingRNase H digestion of the RNA-DNA hybrids and E. coli DNA polymerase I.T4 DNA polymerase was used to polish the blunt-ended cDNA, and thenhemiphosphorylated, Not-1/EcoR-1 adaptors were added by blunt-endligation. A portion (0.3μg) of this cDNA was size-fractionated byagarose gel electrophoresis, and a fraction (300-3000 bp) was recoveredby electroelution. The cDNA was ligated to EcoR-1 digested lambda gt10(Stratagene) and the recombinant phage was packaged using Gigapak-Goldpackaging extract (Stratagene). Approximately 5×10⁶ independent phagewere obtained and 106 phage were plated at a density of 3×10⁴ /150mmplate on a lawn of C600 hfl⁻ E. coli. Duplicate lifts were made usingColony/Plaque Screen filters (DuPont). The filters were screenedsequentially using three probes: 5'-AATCCTGTAAGCTGTGTTAGGAATAAAGGCATCTGTGTGCCGAT-3', SEQ ID NO:10,5'-AATAAAGGCATCTGTGTGCCGATCAGGTGTCCTGGAAGCATGAAACAGATTGG-3', SEQ IDNO:11 and the PCR product, PCR-BT40.1 (sequence not determined, theproduct was derived as detailed under PCR herein). The standardconditions for screening were modified: 50° C., 6× SSC for hybridizationand wash with the oligonucleotide probes; and 37° C., 5× SSC/20%formamide and 55° C, 6× SSC for hybridization and wash, respectively forthe double stranded probe.

Selected clones were plaque purified, and DNA was obtained from liquidlysates. The cDNA inserts were obtained by digestion with EcoRI, andsubcloned into Bluescript plasmid (Stratagene).

Northern blot analysis

RNA was fractionated by agarose gel electrophoresis in the presence offormaldehyde and blotted to nylon membranes (Nytran, Schleicher andSchuell) by the capillary technique. Radioactive labelled DNA probeswere hybridized to the immobilized RNA in 20% Formamide/5×SSC/5×Denhardt's/0.1% SDS at 42° C., and washed in 0.1×SSC/0.1% SDS at 65° C.

Antimicrobial assays

Antimicrobial activity during purification was determined by plate assaydescribed in Zasloff, "Magainins, a class of antimicrobial peptides fromXenopus skin: Isolation, characterization of two active froma, andpartial cDNA sequence of a precursor," Proc Natl Acad Sci, USA,84:5449-53 (1987). A concentrated aliquot of each fraction (2-5ul) wasspotted onto a lawn of E. coli strain D31 (Steiner, H.D., Hultmark, A.et al., "Sequence and specificity of two antibacterial proteins involvedin insect immunity," Nature, 292: 246-248 (1981)) on a petri dishcontaining 10 g/l Bacto Tryptone, 5g/l Yeast extract, 0.75% agarose(Sigma), 25mM Tris, pH7.4, 50 mM NaF, and incubated overnight at 37° C.

Minimal inhibitory concentration (MIC) of the peptide was determined byincubating 2.5×10⁴ bacteria in 0.25× Tripticase Soy Broth (TSB) withincreasing concentrations of the peptide in 96 well microtiter plates(Corning), overnight at 37° C.

RESULTS

Isolation of the peptide

The bovine tracheal epithelium was extracted in acid andsize-fractionated by gel filtration on Biogel P-30 (FIG. 1A). Theelution profile of the tracheal extract showed two peaks of U.V.absorbance (monitored at 220nm). Antibacterial activity was assayedusing E.coli strain D31 (Steiner, H.D. Hultmark, A., et al., supra(1981)) for fractions 13 (void volume) to 60 (included volume);fractions 32-36 showed significant activity, as evidenced by clear zonesof killing (FIG. 1B). These fractions corresponded to the peptide region(i.e., <5,000 Da) when analyzed by SDS-polyacrylamide gelelectrophoresis and silver stained for protein (data not shown).

The antimicrobial fractions were pooled and then fractionated on ionexchange HPLC (FIG. 1C). An isolated peak at 25.6 minutes contained theonly detectable antimicrobial activity. This peak was collected andfurther purified by reverse phase HPLC (FIG. 1D). There was a singlesuperimposed peak of protein (arrow) and antimicrobial activity (datanot shown) eluting at 37.3% CH₃ CN. The overall yield was approximately2μg/g of epithelium (wet weight). The purity of the peptide was 95% asassayed by analytical HPLC and acid-polyacrylamide gel electrophoresis(data not shown) which showed a single, basic band.

Protein sequence analysis of TAP

The purified peptide was subjected to protein sequence analysis usingautomated liquid phase sequencing and amino acid compositional analysis(TableI). The amino-terminal residues were analyzed directly, and themore carboxy-terminal residues were analyzed following cyanogen bromidecleavage (FIG. 2A). The sequence determined by protein sequence analysispredicted a molecular weight of 3443. The molecular weight of thepurified peptide was determined by mass spectroscopy analysis to be4085.5, indicating that the protein sequence was incomplete. It wasdecided to deduce the remainder of the peptide sequence by analysis of acloned cDNA.

                  TABLE I                                                         ______________________________________                                        Amino acid analysis of TAP                                                    Amino acid composition was detrmined after hydrolysis of                      200 pmol of purified TAP (SEQ ID NO:1). Results are                           expressed as mol amino acid per mol protein.                                  Amino Acid    Analysis (sequence)                                             ______________________________________                                        Asp*          2.17 (2)                                                        Asn                                                                           Glu*          0.83 (1)                                                        Gln                                                                           Ser           1.31 (2)                                                        Gly           3.51 (4)                                                        Arg           3.09 (4)                                                        Thr           0.94 (1)                                                        Ala           0.97 (1)                                                        Pro           2.93 (3)                                                        Val           4.59 (5)                                                        Met           1.25 (1)                                                        Cys           3.77 (6)                                                        Ile           2.54 (3)                                                        Lys           3.82 (5)                                                        ______________________________________                                         *Includes the corresponding amide.                                       

Cloning of the TAP cDNA

Degenerate-oligonucleotide primers (SEQ ID NO:6, SEQ ID NO:12 and SEQ IDNO:7) were designed corresponding to amino acids 1-6(BT-40-1 and -2) and21-26 (BT40-3) respectively as shown in FIG. 2B. Sense Primers 1 (SEQ IDNO:6) and 2 (SEQ ID NO:12) had the EcoRI recognition site incorporatedon the 5' end, and antisense primer 3 (SEQ ID NO:7) had the Sstlrecognition site on its 5' end. These primers were used in a PCR usingbovine tracheal cDNA as template DNA and were expected to amplify thenucleotide sequence coding for amino acids 1-26. The principal DNAproduct was 90 bp in length, as expected based on peptide primarystructure and the selected oligonucleotide primers. This indicated thatcDNA encoding the peptide was present in the library, and yielded a DNAtemplate for probe synthesis.

The cDNA library from bovine tracheal epithelium (approximately 10⁶independent lambda gt10 phage) was screened using three different probesin parallel: the PCR product (no sequence, product obtained as per PCRdescribed herein), and two "best-guess" synthetic oligonucleotide probes(SEQ ID NO:10 and SEQ ID NO:11), designed based on the peptide SEQ IDNO:1 (FIG. 2A) using published codon frequency tables. Lathe, R."Synthetic oligonucleotide probes deduced from amino acid sequence data.Theoretical and practical considerations," J MoI Biol 183: 1-12, (1985).Only clones which hybridized with two of the three probes (of whichthere were 15) were considered positive, and 7 of these were taken forfurther analysis. Several positive clones were plaque purified and theinserts were subcloned into Bluescript plasmid (Stratagene). All insertswere of approximately the same size, and DNA sequence analysis wasperformed on one of them. The sequence for the cDNA clone pBT40-4.4, SEQID NO:4, is shown in FIG. 3.

The cDNA sequence of the precursor protein (FIG. 3, SEQ ID NO:4)contains an open reading frame of 64 amino acids in length from thefirst ATG codon (base 35). The deduced amino acid sequence of SEQ IDNO:1 encoded by nucleotides 113-225 of SEQ ID NO:2, beginning with anN-terminal asparagine residue is in perfect agreement with the aminoacid data (FIG. 2D). The open reading frame, which extends 5 residuesbeyond the most carboxy-terminal residue of the sequence elucidated bypeptide analysis, is followed by an in-frame termination codon. Theamino acid sequence of the peptide from this clone has a predictedmolecular weight of 4091; if all cysteine residues are involved indisulfide bonds, this would reduce the predicted molecular weight to4085, in complete agreement with the mass spectroscopic data. The aminoacid composition of the predicted peptide agrees favorably with thepeptide data.

Northern blot analysis

RNA isolated from whole lung and isolated tracheal mucosa was subjectedto northern blot analysis using the cDNA insert (SEQ ID NO:4) as a probe(FIG. 4). Under stringent conditions the cDNA (SEQ ID NO:4) proberecognized an abundant message of approximately 400 bp in bovine tracheamRNA, along with a less abundant species of the same size in bovine lungRNA. Both lanes had identical amounts of RNA as evidenced by ethidiumbromide staining of the gel (data not shown) and by hybridization to abovine alpha-butulin probe (FIG. 4).

Antimicrobial activity of TAP

The purified bovine TAP, obtained as described herein, was tested onseveral strains of bacteria to determine its antimicrobial activity invitro. The results shown in Table II indicate that TAP (SEQ ID NO:1) hasantimicrobial activity for both gram positive and gram negativebacteria. In addition, the peptide has significant activity against thefungus Candida albicans. The observed activity of the peptide, SEQ IDNO:1, is on the same order of magnitude as synthetic Magainin-2-NH₂ (SEQID NO:5), in the assay used here.

                  TABLE II                                                        ______________________________________                                        Antimicrobial activity of TAP                                                 Antimicrobial activity of TAP (SEQ ID NO:1) and                               Magainin 2-NH.sub.2 (SEQ ID NO:5). Minimal inhibitory                         concentrations were determined by incubating approximately                    2.5 × 10.sup.4 microbe in 0.25X TSB with 50, 25, 12.5, 6.25 or          3.125 μg/ml of the appropriate peptide.                                                Minimal inhibitory con-                                                       centration (μg/ml)                                                           Magainin 2-NH.sub.2                                                                         TAP                                               Organism (ATCC)                                                                             SEQ ID NO:5   SEQ ID NO:1                                       ______________________________________                                        Escherichia coli*                                                                           3.125-6.25    12.5-25                                           Klebsiella pneumonia                                                                        3.125-6.25    12.5-25                                           (13883)                                                                       Staphylococcus aureus                                                                       25-50         25-50                                             (25923)                                                                       Pseudomonas aeruginosa                                                                      6.25-12.5     25-50                                             (27853)                                                                       Candida albicans (14053)                                                                    25-50         6.25-12.5                                         ______________________________________                                         *strain D31 (Steiner, H.D., Hultmark, A., supra (1981)   *strain D31          (Steiner, H.D., Hultmark, A., supra, (1981)

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AsnProValSerCysValArgAsnLys GlyIleCysValProIle                                151015                                                                        ArgCysProGlySerMetLysGlnIleGlyThrCysValGlyArg                                 2025 30                                                                       AlaValLysCysCysArgLysLys                                                      35                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 114 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AATCCTGTAAGCTGTGTTAG GAATAAAGGCATCTGTGTGCCGATCAGG48                           AsnProValSerCysValArgAsnLysGlyIleCysValProIleArg                              151015                                                                        TGTCCTGGAAGCATGAAACAGATT GGCACCTGTGTTGGGCGGGCAGTA96                           CysProGlyArgMetLysGlnIleGlyThrCysValGlyArgAlaVal                              202530                                                                        AAATGCTGTAGAAAGAAG 114                                                        LysCysCysArgLysLys                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       M etArgLeuHisHisLeuLeu                                                        25-20                                                                         LeuAlaLeuLeuPheLeuValLeuSerAlaTrpSerGlyPheThrGln                              15-10 -5                                                                      GlyValGlyAsnProValSerCysValArgAsnLysGlyIleCysVal                              1510                                                                          ProIleArgCysProGlySerMetLysGlnIleGlyThrCysValG ly                             152025                                                                        ArgAlaValLysCysCysArgLysLys                                                   3035                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 349 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CCGCGGCCGCCGCCGAGCCGCTCGGGACGCCAGC34                                          ATGAGGCTCCATCACCTGCTCCTCGCGCTCCTCTTCCTGGTCCTGTCT82                            MetArgLeuHisHisLeuLeuLeuA laLeuLeuPheLeuValLeuSer                             25-20-15                                                                      GCTTGGTCAGGATTTACTCAAGGAGTAGGAAATCCTGTAAGCTGTGTT130                           AlaTrpSerGlyPheThrGlnGlyValGlyAsnProV alSerCysVal                             10-515                                                                        AGGAATAAAGGCATCTGTGTGCCGATCAGGTGTCCTGGAAGCATGAAA178                           ArgAsnLysGlyIleCysValProIleArgCysProGlySer MetLys                             101520                                                                        CAGATTGGCACCTGTGTTGGGCGGGCAGTAAAATGCTGTAGAAAGAAG226                           GlnIleGlyThrCysValGlyArgAlaValLysCysCysArgLysLys                               253035                                                                       TAAAAGAAGGCCAAGACACAGCCGGGATCAATGCCCAGTCAGAAACTGCG276                         CCCTTTGACAGAGCGTCTAAAATTTAAACCAGAATAAATTTTGTTCAAAG326                         TTAAAA AAAAAAAAAAAAAAAAA349                                                   (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GlyIleGlyLysPheLeuHisSerAlaLys                                                 1510                                                                         LysPheGlyLysAlaPheValGlyGluIle                                                1520                                                                          MetAsnSer                                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GAATTCAAYCCHGTBTCYTGYGTT24                                                    (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acids                                                       (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GAGCTCDGTNCCDATYTGYTTCAT24                                                    (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                      AACCCTGTCTCCTGTGTGCGCAACAAGGGCATCTGTGTGCCCAT44                                (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        AACAAGGGCATCTGTGTGCCCATCCGCTGCCCTGGCTCCATGAAGCAGATTGG53                      (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AATCCTGTAAGCTGTG TTAGGAATAAAGGCATCTGTGTGCCGAT44                               (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AATAAAGGCATCTGTGTGCCGATCAGGTGTC CTGGAAGCATGAAACAGATTGG53                      (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GAATTCAAYCCHGTBAGNTGYGTT 24                                               

We claim:
 1. A substantially pure, peptide essentially comprising theamino acid sequence defined in SEQ ID NO:1.
 2. A substantially pure,peptide essentially comprising the amino acid sequence defined in SEQ IDNO:3.
 3. A protein produced from the cDNA encoding an antimicrobialpeptide isolated from mammalian tracheal mucosa, wherein said cDNAessentially comprises the DNA sequence defined in SEQ ID NO:2.
 4. Aprotein produced from the cDNA encoding a precursor antimicrobialpeptide isolated from mammalian tracheal mucosa, wherein said cDNAessentially comprises the DNA sequence defined in SEQ ID NO:
 4. 5. Asubstantially pure, isolated mammalian precursor antimicrobial peptidecomprising a protein having substantially the same amino acid sequenceas the protein defined in SEQ ID NO:3.
 6. A substantially purified,isolated mammalian antimicrobial peptide comprising a protein havingsubstantially the same amino acid sequence and at least the sameantimicrobial activity as the protein defined in SEQ ID NO:1.
 7. Thesubstantially purified isolated mammalian precursor antimicrobialpeptide of claim 5 wherein the mammalian source is bovine.
 8. Thesubstantially purified isolated mammalian antimicrobial peptide of claim6 wherein the mammalian source is bovine.
 9. An antimicrobial peptideproduced from a recombinant expression vector comprising a DNA sequencesubstantially the same as the DNA sequence defined in SEQ ID NO:2. 10.An antimicrobial peptide produced from a recombinant expression vectorcomprising a DNA sequence substantially the same as the DNA sequencedefined in SEQ ID NO:4.